Identification of in vivo disulfide conformation of TRPA1 ion channel

Abstract

TRPA1 (transient receptor potential ankyrin 1) is an ion channel expressed in the termini of sensory neurons and is activated in response to a broad array of noxious exogenous and endogenous thiol-reactive compounds, making it a crucial player in chemical nociception. A number of conserved cysteine residues on the N-terminal domain of the channel have been identified as critical for sensing these electrophilic pungent chemicals, and our recent EM structure with modeled domains predicts that these cysteines form a ligand-binding pocket, allowing for the possibility of disulfide bonding between the cysteine residues. Here, we present a comprehensive mass spectrometry investigation of the in vivo disulfide bonding conformation and in vitro reactivity of 30 of the 31 cysteine residues in the TRPA1 ion channel. Four disulfide bonds were detected in the in vivo TRPA1 structure: Cys-666-Cys-622, Cys-666-Cys-463, Cys-622-Cys-609, and Cys-666-Cys-193. All of the cysteines detected were reactive to N-methylmaleimide (NMM) in vitro, with varying degrees of labeling efficiency. Comparison of the ratio of the labeling efficiency at 300 μM versus 2 mM NMM identified a number of cysteine residues that were outliers from the mean labeling ratio, suggesting that protein conformation changes rendered these cysteines either more or less protected from labeling at the higher NMM concentrations. These results indicate that the activation mechanism of TRPA1 may involve N-terminal conformation changes and disulfide bonding between critical cysteine residues.

FIGURE 1.

Two-dimensional schematic representation of a TRPA1 dimer. The cytoplasmic N and C termini are separated by six transmembrane helices (red arrows), which form the central pore. The monomer contains 31 cysteine residues (blue circles), and the N terminus has an extended ankyrin repeat domain (gray box).

FIGURE 2.

MS of in vivo disulfide bonds and in vitro labeling of TRPA1 cysteines. A, high accuracy FT mass spectrum of the triple-charged precursor ion formed by peptides 663–672 and 622–626 (m/z 602.2715³⁺), allowing the assignment of a Cys-666–Cys-622 disulfide bond. Sequence information is shown for the b and y ions of chain A (Ab₂–Ab₄ and Ay₃–Ay₉) and chain B (Bb₂–Bb₄ and By₂–By₄). The mass difference of 708.228 Da between the double-charged y ion peaks Ay₆²⁺ (m/z 346.686) and Ay₇²⁺ (m/z 700.800) matches the mass of the sequence C-s-s-CPIM_oxE (708.2281 Da) within 0.02-Da mass tolerance. Similarly, the mass difference between single-charged b ions Ab₃ (m/z 405.2131) and Ab₄ (m/z 1113.4392) is 708.226 Da. B and C, extracted chromatographic peak of peptide 663–672 for Cys-666 labeled with IA (B) or with NMM (C). The fragmentation patterns for the peptides are identical, aside from the expected mass shifts of the product ions starting from y₇ (insets).

FIGURE 3.

In vitro reactivity of TRPA1 cysteines to NMM. A, fractional reactivity of TRPA1 cysteines with 300 μm NMM (gray bars) and 2 mm NMM (black bars). Labeling was calculated in triplicate by dividing the peak area of the NMM-labeled peptides by the sum of the peak areas for IA-modified and NMM-modified peptides (NMM_modified/total_modified). B, histogram of the low [NMM] to high [NMM] ratios. The fractional reactivity of the cysteine at 300 μm NMM was divided by its fractional reactivity at 2 mm NMM. The average ratio of reactivity was 0.864 (solid vertical line) with an S.D. of 0.185 (dashed vertical lines). Outliers from the mean are labeled.

FIGURE 4.

EM density and N-terminal model of TRPA1 showing cysteines involved in disulfide bonds. An I-TASSER-generated model of the N terminus of TRPA1 (blue ribbon) fit into the EM density reconstructed previously (see Ref. 12). The cysteines involved in disulfide bonding are labeled and are displayed in orange.